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Cardiovascular Research 58 (2003) 369–377
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Review
Stem cells and cardiac disorders: an appraisal
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Michael J. Goldenthal, Jose´ Marın-Garcıa
The Molecular Cardiology and Neuromuscular Institute, 75 Raritan Avenue, Highland Park, NJ 08904, USA
Received 28 August 2002; accepted 12 November 2002
Abstract
The use of stem cells has proved to be an important tool in investigating the events of early cardiac development, differentiation, and
morphogenesis. In addition, stem cell transplantation in the treatment of certain cardiac disorders has shown early promise. We have
attempted to present a balanced review of both basic studies and clinical–therapeutic potential of stem cells transplantation in the
damaged heart.
 2003 European Society of Cardiology. Published by Elsevier Science B.V. All rights reserved.
Keywords: Stem cells; Myocytes; Mitochondria; Endothelial receptors; Ischemia
1. Basic studies
1.1. Cardiomyocyte differentiation of embryonic stem
cells
Embryonic stem cells (ES) are valuable models to study
the events of vascular morphogenesis, cardiac growth and
differentiation and cardiac disorders. The delineation of
early events in development and differentiation which are
extremely difficult to assess in the mammalian heart are
possible in ES cells. These studies have allowed progress
in the identification and characterization of regulatory
factors, elucidation of the pathways of transcriptional
activation and the signal transduction events involved in
cardiomyocyte differentiation.
Pluripotent ES cells can spontaneously differentiate, via
embryo-like aggregates termed embryoid bodies, into
early-stage cardiomyocytes in vitro by manipulation of the
culture growth medium [1–4]. These cardiomyocytes can
be of the pacemaker-atrium and ventricle-like type, being
distinguishable by their specific patterns of action potentials [4,5]. In vitro cardiomyocyte differentiation has
been established with both murine [1,3–5] and human [2]
ES cells. To maintain their pluripotentiality, both mouse
*Corresponding author. Tel.: 11-732-220-1719; fax: 11-732-2202992.
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E-mail address: [email protected] (J. Marın-Garcıa).
and human embryonic stem cells require cultivation on
feeder layers (usually mouse fibroblast cells). Interestingly,
the addition of a differentiation inhibiting cytokine i.e.
leukemia inhibitory factor (LIF) can replace the feeder
cells with mouse but not human ES cells [6]. The role that
LIF plays in modulating ES cell and cardiomyocyte
differentiation is dependent on the developmental stage.
For instance, it can hamper early events in cardiomyocyte
differentiation while promoting proliferation at more fully
differentiated states [7]. It is also important to note that
cardiomyocyte levels are increased in ES cultures supplemented with specific growth factors (e.g. EGF and
retinoic acid) but that none of these factors direct ES cell
differentiation to a single cell type [8]; cardiomyocytes can
be subsequently purified from a heterogenous mixture of
ES-derived cells.
Retinoic acid (RA) has been found to effect the efficiency of cardiomyocyte differentiation in a time and
concentration dependent manner [4]. In vitro studies with
mouse ES cells demonstrated that RA can specifically
induce the formation of ventricular-specific cardiomyocyte
[4], increasing cardiomyocyte number from ES cells and
specifically enhanced ventricular cell differentiation albeit
the number of pace-maker and atrial cells were reduced. In
contrast, increasing RA concentration or RA treatment at
the initial stages of embryoid body development (days
Time for primary review 27 days.
0008-6363 / 03 / $ – see front matter  2003 European Society of Cardiology. Published by Elsevier Science B.V. All rights reserved.
doi:10.1016 / S0008-6363(02)00783-6
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1–5) has been reported to inhibit cardiogenesis [4] and
induces the formation of neuronal and glial cells from
pluripotent ES cells and embryoid bodies in parallel with
increased activity of PAX6, a transcription factor involved
in central nervous system development [9]. RA is pivotally
involved in early cardiac development with both positive
and negative aspects and RA deprivation can induce
cardiac malformations. Also, defective cardiac development has been found in association with increased levels of
RA treatment (RA toxicity) with marked effect on expression of cardiac specific transcriptional factors [10,11]. RA
primarily exerts its effect on development and cellular
differentiation through the nuclear retinoic acid receptors
(e.g. RXR) that modulate the transcription of an array of
specific target genes by binding specific DNA sequences.
Recently, Nemer et al. reviewed the role that cardiac
specific transcription factors plays in cardiomyocyte differentiation and cardiac development [12]. The expression
of cardiac gene products occurs in a developmentally
controlled program both in early myocardial development
in vivo and in ES cell-derived cardiomyocytes. In both
early embryogenesis and in early ES cells, expression of
the cardiac specific transcription factors GATA-4, NKX
2.5 and members of the myocyte enhancer family (MEF2), e.g., MEF2c precedes (and mediates in turn) the
activation of expression of markers of early, intermediate
and terminal cardiac cell differentiation including atrial
natriuretic factor (ANF) and B-type natriuretic peptide
(BNP), myosin light chain (MLC), a-myosin heavy chain,
b-myosin heavy chain, and cardiac troponin C. Blocking
specific GATA-4 transcription (with antisense transcripts)
eliminates the formation of beating cardiac muscle cells
and abolishes transcription of specific cardiac markers
significantly reducing levels of MEF2c and NKX2.5.
Although GATA-4 is one of the first cardiac transcription
factors to be induced and plays a key role in cardiomyocyte differentiation, both loss-of-function and gainof-function experiments have demonstrated that GATA-4
is not sufficient for the initiation of cardiac differentiation.
ln the absence of GATA-4, embryonic stem cells can
differentiate into mesoderm and can initiate the cardiogenic pathway but are unable to proceed beyond a
precardiac (cardioblast) stage [12]. Similarly, while
GATA-4 overexpression in embryonic stem cells potentiates cardiogenesis, its stimulatory effect on cardiac
differentiation requires cell aggregation and the presence
of other interacting factors [13]. Another important transcriptional activator, the cardiac homeodomain-containing
NKX2.5 which is expressed in cardiac progenitor cells at
early development, likely acts downstream of GATA-4
(since inactivation of NKX2.5 does not affect GATA-4
expression) and may play a role in late cardiac differentiation events (Fig. 1). It is important to note that both
GATA-4 and NKX2.5 work in combination with other
transcription factors; for instance, GATA-4 recruits MEF2
proteins to GATA binding sites to activate synergistically
the promoters of several critical genes involved in both
cardiac differentiation and in maintaining the cardiac
phenotype during postnatal development including MLC,
ANF, a-myosin heavy chain, desmin, troponin T and I and
a-actin [14]. While MEF2c is likely downstream of both
GATA-4 and NKX2.5, in mouse embryos containing null
alleles of MEF2c, cardiomyocyte differentiation was disrupted, the ventricle failed to form and a subset of cardiac
muscle genes (including ANF) were not expressed [15].
While these cardiac transcription factors have been the
focus of much research, the mechanism by which these
critical transcription factors themselves are regulated remains elusive. Moreover, several growth factors have been
identified which play an important role in the initial
induction of cardiac differentiation, e.g., bone morphogenetic proteins [16]. However, the precise pathway of
the molecular cascades stemming from these growth
factors leading to cardiac-specific gene expression (required for cardiac specification) remains to be elucidated.
Molecules that are expressed in precardiac cells preceding
the cardiac-specific transcription factors will be soon
identified using such molecular techniques as differential
display and microarray analysis allowing better definition
of the molecular mechanism by which induction of cardiac
differentiation is controlled.
Investigation of the developing mammalian cardiomyocyte’s signaling cascade in response to b-adrenergic stimulation has compared ES cell response from
early (embryoid bodies) and later developmental stages of
the differentiating ES cells. The calcium current (measured
by patch-clamp methodology) in early ES-derived cardiomyocytes were insensitive to isoproterenol, forskolin
and 8-bromo-cAMP. Electrophysiological characteristics in
late developmental stage of ES-cell derived cardiomyocytes were identical to postnatal cardiomyocytes
suggesting that the signalling cascade components have
become functionally coupled during cardiomyocyte development [17]. The hypo-responsivity of early cardiomyocytes to b-adreneregic agonists is especially
noteworthy since a variety of cardiac disorders, including
heart failure, are characterized by depressed function of
b-adrenoreceptors and deficient production of cyclic AMP
while altered G protein expression and coupling have been
reported in the hypertrophic heart. In addition, it is wellknown that both heart failure and cardiac hypertrophy are
associated with a re-expression of the fetal gene program.
However, recent evidence demonstrated that ES-derived
cardiomyocytes manifest increased arrhythmogenic potential with marked heterogeneity in the initiation, duration
and spontaneous activity of the action potential in contrast
to adult myocytes [18], a caution to be considered in their
potential use as donor cells in transplantation.
Interestingly, increased levels of oxidative stress can
impact on cardiomyocyte development from embryoid
bodies [19], e.g., Reactive oxygen species (ROS) is
generated in increased quantities during embryoid body
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Fig. 1. Pathways leading to the differentiation of the cardiac cell. Shown are events at the plasma membrane (include growth and morphogenetic factor
receptors and the signaling apparatus including the b-adrenergic system), the generation of reactive oxygen species (ROS) at the mitochondria and its
impact on downstream events. Also depicted is the centrally located cardiomyocyte nucleus with its cascade of master regulatory transcription factors (e.g.
GATA-4, NKX2.5 and MEF2c) and the activation of gene expression involved in the cardiomyocyte phenotype including connexin (major protein of the
gap junction), ANF, BNP, sarcomeric and contractile prtoeins.
development, and incubation with the pro-oxidants H 2 O 2
or menadione was found to enhance cardiomyogenesis.
Similarly, reduced ROS levels resulting from addition of
free radical scavengers significantly decreased the number
of beating embryoid bodies. The role of ROS in ES
cardiomyogenesis is consistent with recent data indicating
that a rapid and significant increase in intracellular ROS is
associated with growth factor or cytokine stimulation [20].
Phosphatidylinositol 3-kinase (PI-3-kinase) plays a pivotal
role in the signaling and regulation of intracellular ROS
levels and cardiomyogenesis [19]. The number of beating
embyroid bodies was significantly reduced in parallel with
decreasing ROS levels when ES cells are treated with
LY294002, a specific inhibitor of PI-3-kinase. Raising the
ROS levels in the presence of the PI-3-kinase inhibitor (by
adding pro-oxidants) restored the levels of cardiomyocyte
differentiation.
Also, it is important to note the relatively easy genetic
manipulation of ES cells in constructing gene knock-out
lines. This facilitates the examination of the functional role
of specific targeted genes on cardiomyocyte differentiation
and function without generating transgenic animals or as a
critical intermediate in their generation.
1.2. Pluripotent embryonic carcinoma cell-lines can
differentiate into cardiomyocytes
The pluripotent mouse embryonal carcinoma cell line
P19 is a well-characterized model of cell differentiation
and can give rise to the formation of all three germ layers
thought to differentiate by the same mechanisms as normal
embryonic ES. These cells ressemble the inner mass of the
blastocyst and their differentiation mimics early events of
embryogenesis. Under appropriate culture conditions,
pluripotent P19 cell line can differentiate via embryo-like
aggregates into spontaneously beating myocytes. In addition, clonal derivatives of P19 cells (PL19CL6) have been
isolated which are more efficient at cardiomyocyte differentiation [21].
Several studies have utilized treatment with the solvent
dimethyl sulfoxide (DMSO) to induce cardiac differentiation of P19 cells presumably through the activation of
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essential transcription factors (GATA-4 and Nkx-2.5).
Exposure of P19 cells to RA leads to neural cell differentiation and selective endothelin receptor (ET B ) expression
[22]. In contrast, cardiomyocytes derived from this cell
line have a pronounced induction of another class of
endothelin receptor (ETA ) which lead to increases in ANF
levels. Recently, oxytocin was found to be a potent inducer
of P19 cardiomyocyte differentiation [23] albeit how this is
achieved remains to be defined. P19 derived cardiomyocytes exhibit action potentials similar to embryonic
cardiomyocytes with full functional expression of adrenoceptors and calcium channels [24].
The opportunity to identify specific factors that mediate–promote cardiomyocyte differentiation has been pursued with P19 cells using differential display. A unique
transcript and its gene have recently been characterized
(Midori) whose gene product is developmentally regulated
in the heart. Overexpression of Midori increased the
efficiency of cardiomyocyte differentiation by P19 cells
supporting its role in mediating that process [25].
2. Stem cell-transplantation studies: myocardial
repair and treatment of cardiac disorders
2.1. Cardiomyocyte transplants
It is generally agreed that the adult heart lacks the ability
to regenerate. While there is recent evidence that a very
limited proportion of cardiac cells maintain the ability to
divide [26], the ability to easily grow cardiomyocyte cells
in culture in quantities large enough to repair injured
myocardium appears to be extremely limited. Recent study
has suggested that the limitations of cardiomyocyte proliferation may ultimately be reversed by removing the cellcycle block thereby allowing terminally differentiated
cardiac cells to re-enter the cell cycle [27]. Early experiments with fetal cardiomyocyte transplantation showed
success in the formation of stable grafts and nascent
intercalated discs between grafted and host myocardial
cells [28,29]. Since the cardiomyocytes used in the majority of successful transplantation studies have been of fetal
origin, questions have been raised as to the extent of in
vivo differentiation of the implanted cells into mature
myocardium [30]. Investigation of the fate of neonatal rat
cardiomyocytes implanted from male donors into normal
female hearts as gauged by quantitative PCR analysis of
the male-specific Sry gene showed that by 1 h after
transplant, there was a considerable loss of donor cells at
the graft site (57%) which decreased further to 24% over
24 h and to 15% over 12 weeks [31]. This study not only
suggested the presence of substantial cell death after
transplant, but also indicated that the techniques for cell
injection need further optimization given the striking
variability of graft efficiency. In addition, several investigators have addressed the concern that cardiomyocyte
transplantation also may cause further problems at the site
of myocardial grafting unless high levels of cardiomyocyte
cell death (presumably due to ischemia) are avoided
[32,33]. The demonstration of markedly increased cell
death (using TUNEL analysis) at the graft site as well as
the finding that no increase in graft size occurs with
increasing number of injected cardiomyocytes [32] have
prompted careful consideration of the clinical use of
cardiomyocyte transplantation in treating the ischemic
heart. In this regard, increased research focus is needed to
develop successful strategies that can maximize grafted
cardiomyocyte cell survival and enhance the differentiation
process (both in vitro and in vivo).
2.2. Skeletal myoblast transplant
There is ample evidence that cells of transplanted
skeletal muscle cells can differentiate and develop into
striated cells within the damaged myocardium, and prevent
progressive ventricular dilatation by improving heart function [34–37]. Skeletal muscle cells have been successfully
delivered to myocardium by either intramural implantation
or arterial delivery [35,36]. Skeletal muscle satellite cells
can proliferate abundantly in culture, and can be easily
grown from the patient themselves (self-derived or autologous) thereby reducing a potential immune response.
Importantly, skeletal myoblasts are relatively ischemiaresistant (compared to cardiomyocytes) since they can
withstand several hours of severe ischemia without becoming irreversibly injured as compared to cardiomyocytes
(which injure rapidly within 20 min) [38]. Potential
limitations in the use of skeletal muscle include the
unknown myoblast cell survival, in situ myocardial integration (e.g. electrophysiologically), the stability of differentiated phenotype and long-term fate of the transferred
myoblasts and the extent of myocardial functional benefits
(e.g. contractility) with their transplant. Another limiting
factor in the transplantation of skeletal myoblasts relates to
the necessary concentration of cells delivered, (too much
may be as problematic as too little) and the timing of
growth (relative to the myocardial injury). In addition, in
the repair of large myocardial infarct, putting new cells at a
site of limited blood–nutrient–oxygen supply may (in
some cases) not be sufficient for myocardial repair.
There is evidence that the cardiac environment is not
only permissive for myogenic differentiation, but also can
influence the developmental program of implanted myoblasts enabling them to better assist cardiac performance
[35,39]. When neonatal skeletal myoblasts were transplanted into injured rat hearts, a gradual establishment of
slow-twitch, fatigue-resistant muscle fiber phenotype in
implanted cells occurred (with physiological characteristics
similar to cardiac muscle including high OXPHOS capacity, fatigue resistance and use of b-MHC as a major
contractile protein. Therefore, when grafted into injured
hearts, skeletal myoblasts could establish new muscle
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tissue which contracted when electrically stimulated. Two
decades ago, Menasche found that the latissimus dorsi
skeletal muscle undergoes fiber type switching when
conditioned for dynamic cardiomyoplasty. When conditioned by repeated electrical stimulation, there was a
conversion from fast fiber–easy fatigability to slow fiber
phenotype [37].
In addition, adult skeletal muscle contains a population
of cells with several characteristics of bone-marrow hematopoietic stem cells [40]. However, Murry et al. and
Reinecke et al. have not been able to observe the differentiation of skeletal muscle myoblasts to cardiomyocytes
after cardiac grafting [39,41]. These authors conclude: (1)
that skeletal satellite muscle cells injected into the heart
differentiate into mature skeletal muscle and do not
express cardiac-specific genes after cardiac grafting; (2)
the demonstration of ‘trans-differentiation’ of skeletal
muscle to cardiomyocyte was either ambiguous in defining
cell lineage or not rigorous when using definitive characteristics to identify the cardiomyocyte phenotype.
2.3. Other ‘ sources’ for stem cell transplant
2.3.1. Bone marrow cells
These multipotent stem cells are extremely responsive to
their microenvironment for milieu-dependent differentiation [42–46]. Infarcted cardiac tissue regenerates with
bone marrow cell (BMC) transplantation. Bone marrow
stem cells have been found to differentiate to cardiomyocytes with 5-azacytidine treatment [42,43]. Signal
transduction is markedly effected with BMC differentiation; qualitative and quantitative differences are found in
the expression of adrenergic / muscarinic receptors in bone
marrow-derived cardiomyocytes after exposure to 5azacytidine [42].
Bone marrow hematopoietic stem cells can differentiate
into cardiomyocytes, endothelium and smooth muscle
when injected into ischemic ventricle [44,45]. Moreover,
the growth response involving BMC interfered with ventricular scarring and stimulated some development of
vascular structures within the infarcted area [45]. Albeit
regarded as a major breakthrough, one major limitation of
this study is that it was conducted with the mouse heart
(which have limited size infarcts compared to human
myocardium). Since marrow stromal cells can be easily
obtained by bone marrow aspiration and also can proliferate in culture before being used as autologous implants,
their use as a source for stem cell transplants appears to be
both advantageous and promising [46].
A cardiomyogenic cell-line has been generated from
marrow stromal cells grown in vitro in the presence of
5-azacytidine which express cardiomyogenic phenotypes
(capable of forming myotubes that develop synchronous
beating, producing ANF and BNP, staining with antiactinin and anti-desmin antibodies, exhibiting a ventricular–like action potential and evidence of cardiomyocyte-
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like ultrastructure) [47]. These cells could be transplanted
to myocardium in vivo replacing native cardiomyocytes
lost by necrosis or apoptosis. BMCs from adult rats (after
induction to differentiate into cardiomyocytes with in vitro
growth in the presence of 5-azacytidine) were autologously
transplanted into myocardial scar tissue. Transplantation of
BMCs inhibited the ventricular scar from thinning, minimized ventricle dilatation and improved myocardial function [43]. The transplanted BMCs also induced angiogenesis which may contribute to the long-term survival of
the transplanted cells in the scar. Recruitment of BMCs has
also been examined in adult mdx mouse heart, a model of
Duchenne’s muscular dystrophy [48]. Dystrophic female
mdx mice receiving BMC transplant from normal male
mice showed incorporation of donor BMCs in heart and
skeletal muscle as well as the presence in myocardium of
single cardiomyocytes with donor nuclei derived from the
BMCs. While this data is consistent with the growing body
of evidence in support for the plasticity of adult stem cells,
neither the relative number or the percentage of donor cells
detected in the host myocardium nor the functional effects
of the transplant were provided in this study.
Human mesenchymal stem cells derived from adult bone
marrow can undergo in situ myogenic differentiation to a
cardiomyocyte phenotype once transplanted into an adult
heart [46,49]. In addition, human mesenchymal stem cells
transplanted into fetal lamb (in utero) can engraft in
multiple tissues, and can differentiate into cardiac and
skeletal myocytes which can persist for over 1 year [50].
Recent studies have demonstrated that progenitor stem
cells of extra-cardiac origin can be incorporated into the
human heart [51,52]. Some of these cells are found near or
at the site of injury and might be involved in myocardial
repair. These studies have utilized female hearts transplanted into male patients and found evidence of Y
chromosome-containing cardiomyocytes; however the estimated levels of Y chromosome-containing cardiomyocytes
differ substantially in quantity, by over three orders of
magnitude (0.04% compared to 18%). Needless to say,
methods to rigorously detect and to accurately quantitate
cardiac cell populations of extra-cardiac origin are necessary. Defining the precise origin of the cardiomyocytes is
also complicated by the recent finding that bone marrow
cells can readily and spontaneously fuse with existing
myocytes essentially adopting their phenotype (while
retaining the BMC nuclear genome) [53].
2.3.2. Endothelial cells
Endothelial cells derived from embryonic stem cells
from dorsal aorta and from differentiated cells of the
human mmbilical vein can ‘trans-differentiate’ into beating
cardiomyocytes when co-cultured with neonatal cardiomyocytes or when injected into postischemic adult
mouse heart [54]. Adult tissues (e.g. neural stem cell and
lung cells) showed no such capability. This study demonstrated activation of cardiac-specific genes (e.g. cardiac
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specific troponin), development of aligned sarcomeres,
electrical coupling and in vivo differentiation when injected into post-ischemic ventricles. Cell-to-cell contact
with neonatal cardiomyocytes is necessary for this process
since conditioned medium does not support this differentiation. Another important finding from this study was that
cellular signals that induce myocardial differentiation in
endothelial cells are different than those that activate
cardiogenesis in the early embryo. Fibroblast growth factor
(FGF2) and bone morphogenetic protein (BMP4) which
have been implicated in the activation of cardiogenesis in
embryonic heart do not activate myocardial ‘trans-differentiation’ with endothelial cells. The nature of the activating
signals for endothelial cell trans-differentiation to cardiomyocytes has not yet been elucidated. The finding that
neural stem cells from brain showed little capability in
‘trans-differentiating’ into cardiomyocytes was somewhat
unexpected given that previous reports established that
neural stem cells from adult brain can give rise to liver,
blood skeletal muscle and myocardium [55].
defective mtDNA genome has been entirely eliminated by
treatment with ethidium-bromide, and replaced by entirely
wild-type mtDNA genes. In a similar vein, treatment of a
patient’s nuclear DNA defects would involve either specific genetic replacement of the nuclear gene defect (sitespecific homologous recombination can be readily undertaken in ES cells) or if the precise site of the nuclear defect
is not known, by replacement of the entire nucleus of the
patient with a wild-type nucleus. In the near future ES cells
therapy may be used in the treatment of mitochondrialbased cardiac diseases.
Stem cells could also be readily employed in the
pharmacological testing and evaluation of cardiotoxic
compounds [58–60]. As an alternative to in vivo studies, a
test utilizing the differentiation of ES cells into cardiomyocytes (to test chemical toxicity in vitro) has been
developed. Interestingly, RA was strongly embryotoxic
inhibiting cardiac cell differentiation at very low concentrations (several orders of magnitude) compared to the
levels needed to exert cytotoxic effects on the viability of
the ES cells [59].
2.4. Methods and site of delivery of transplanted cells
While we have previously noted that transplanted skeletal myoblasts can be effectively introduced to the heart by
either direct intramural injection or by arterial (usually
coronary) delivery, the majority of studies surveyed in this
review (employing BMC, mesenchymal, endothelial and
cardiomyocytes) used direct injection into the ventricular
wall. While a subset of these studies examined transplanted
cells in normal heart tissues, a number also investigated
transplantation into ischemic cardiac tissues, the majority
employing injection of donor cells into the center of the
ischemic region or scar. It remains to be seen whether this
site impacts on the subsequent loss or retention and
differentiation of the donor cells.
3. Applications of stem cells in mitochondrial defects
and toxicology
Presently, information concerning mitochondrial structure and function in ES cells is limited, nor has the use of
ES therapy been applied to treating cardiac diseases with
extensive mitochondrial enzyme or DNA (mtDNA) abnormalities or for the testing of mitochondrial-based cardiotoxicity.
Cardiac disorders with a pronounced mitochondrialbased cytopathy and bioenergetic dysfunction have been
reported. A subset of these have a genetic basis due to
either defects in mtDNA or nuclear DNA [56]. Recently, it
has been proposed to introduce mtDNA-repaired ES cells
into a patient harboring a mtDNA mutation, thereby
potentially transforming a diseased myocardium into a
healthy one [57]. The mtDNA-repaired cells can be
derived from the patient’s own cells, whose endogenous
4. Conclusions
While both embryonic and adult stem cells have shown
exciting possibilities in their projected use in cardiac
transplantation and repair, at present there are limitations
in their use (Table 1). In addition to the difficulties
presently raised by significant ethical, legal and distribution issues with the use of embryonic cells, there are
technical challenges that will have to be surmounted. Both
embryonic and adult stem cells generate heterogenous
populations of cells of which cardiomyocytes represent a
small fraction [61]. It has been estimated that millions of
cardiac cells would be required to repopulate and repair a
single damaged human heart. The ability to direct specific
cardiomyocyte differentiation (with either adult or embryonic cells) will require further knowledge of the
signaling and transcriptional events involved in cardiomyocyte differentiation. The promising developmental
plasticity of adult stem cells (and of multipotent adult
progenitor cells (MAPC) that co-purify with mesenchymal
stem cells [62]) may allow circumvention of the ethical
and availability issues currently associated with embryonic
cell studies as well as providing an autologous source of
highly proliferative cells bypassing the issues of immune
rejection (which could arise with embryonic stem or
cultured cells of heterologous origin).
In addition, rigorous criteria are needed in further
studies of the efficacy of cell transplantation, effects on
stability and function of the transplanted cells and in the
unequivocal identification of transplanted cells. To evaluate the most effective sources of transplanted cells, the
following are necessary:
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Table 1
Myocardial cell transplants: advantages and limits associated with cell-type
Cell-type
Advantages
Limitations
Refs.
Cardiac cells
1. Recognition of myocardial
growth factors and
recruitment to myocardium
are likely faster and more
efficient than other cell-types.
2. In vivo electrical coupling
of transplanted cells to
existing myocardiium has
been demonstrated
1. Poor cell growth in vitro.
2. Transplanted cells are
very sensitive to ischemic
insult and apoptotic cell
death.
3. Fetal rather than adult
source used.
[28–33]
Skeletal myoblast
1. Cells proliferate in vitro
(allowing for autologous
transplant).
2. Ischemia-resistant.
3. Transplanted myoblasts can
differentiate into slow-twitch
myocytes (similar to
cardiomyocytes) enabling
cellular cardiomyoplasty.
4. Reduces progressive
ventricular dilatation and
improves cardiac function.
5. Can use adult cells.
1. Likely do not develop
new cardiomyocytes in
vivo.
2. Electrical coupling to
surrounding myocardial
cells is unclear.
3. Long-term stability of
differentiated phenotype
unknown.
[34–41]
Bone–marrow
stem cell
1. Pluripotent stem cells can
develop into cardiomyocytes.
2. Stem cells are easy to
isolate and grow well in
culture.
3. Neovascularization can also
occur at the site of myocardial
scar which may diminish
ischemia.
4. Can improve myocardial
function.
1. New program of cell
differentiation program is
required.
2. Efficiency of
differentiation into adult
cardiomyocytes (rather
than fetal) appears
limited.
[42–49]
Endothelial cells
1. Transdifferentiation of cells
into cardiomyocyte in vivo
has been shown.
1. New program of cell
differentiation is required.
2. Efficiency, signaling,
stability and regulation of
differentiation unknown.
[54]
Embryonic stem cells
carcinoma cell line
(P19)
1. Easy propagation and
well-defined cardiomyocyte
differentiation process.
1. Potential for tumor
formation and immune
rejection.
2. Incomplete response
to physiological stimuli.
[21]
1. A careful analysis of isoforms of contractile protein
genes, such as of myosin and alpha-actin comparing
their phenotype with that of fetal ventricular cardiomyocytes.
2. Expression of cardiac transcription factors (e.g. Nkx2.5,
eHAND, dHAND, GATA-4) and examination of both
the timing and interaction of cardiac-specific transcription factors in the cascade pathway (Fig. 1).
3. Immunocytological and ultrastructural analyses to examine whether the selected cardiomyocyte cultures are
highly differentiated and evaluating the proportion of
cells which are cardiomyocyte and fibroblast as well as
gauging their origin (double-labelling experiments very
effective).
4. Analysis of specific ion currents and action potentials
characteristic of specific cardiomyocyte populations.
Regenerated cardiomyocytes need to be capable of
responding to appropriate adrenergic and muscarinic
stimulation.
Finally, it is important to underscore that the expression
of specific transcription factors and structural markers of
cardiac differentiation does not represent proof of functionality. Establishing the best cell type for transplantation
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may require refinement of our imaging technology, as well
as further studies to document the efficiency in the
production of cardiomyocytes and their stability and
functioning in the myocardium. It would seem prudent to
remember that the primary objective of the cell transplantation process is to incorporate cells into myocardium
that can perform cardiac work, respond appropriately to
adjacent cardiomyocytes and be appropriately responsive
to physiological stimuli. Also, studies addressing what
kinds of myocardial injury are most likely to be effectively
treated by stem cell transplantation and which are least
likely are necessary. Approaches to cell transplantation
therapy to treat ischemic myocardium must take into
account the rapid death of ischemic tissue and in cases of
myocardial infarction, the size of infarct. Whether results
obtained in animal models of ischemia, cryoinjury or
cardiomyopathy will be reproducible within the setting of
human coronary artery occlusion and extensive myocardial
infarction remains to be seen.
[12]
[13]
[14]
[15]
[16]
[17]
[18]
References
[1] Doevendans PA, Kubalak SW, An RH, Becker DK, Chien KR, Kass
RS. Differentiation of cardiomyocytes in floating embryoid bodies is
comparable to fetal cardiomyocytes. J Mol Cell Cardiol
2000;32:839–851.
[2] Kehat I, Kenyagin-Karsenti D, Snir M, Segev H, Amit M, Gepstein
A, Livne E, Binah O, Itskovitz-Eldor J, Gepstein L. Human
embryonic stem cells can differentiate into myocytes with structural
and functional properties of cardiomyocytes. J Clin Invest
2001;108:407–414.
[3] Doetschman T, Shull M, Kier A, Coffin JD. Embryonic stem cell
model systems for vascular morphogenesis and cardiac disorders.
Hypertension 1993;22:618–629.
[4] Wobus AM, Kaomei G, Shan J, Wellner MC, Rohwedel J, Ji G,
Fleischmann B, Katus HA, Hescheler J, Franz WM. Retinoic acid
accelerates embryonic stem cell-derived cardiac differentiation and
enhances development of ventricular cardiomyocytes. J Mol Cell
Cardiol 1997;29:1525–1539.
[5] Muller M, Fleischmann BK, Selbert S, Ji GJ, Endl E, Middeler G,
Muller OJ, Schlenke P, Frese S, Wobus AM, Hescheler J, Katus HA,
Franz WM. Selection of ventricular-like cardiomyocytes from ES
cells in vitro. FASEB J 2000;14:2540–2548.
[6] Boheler KR, Czyz J, Tweedie D, Yang HT, Anisimov SV, Wobus
AM. Differentiation of pluripotent embryonic stem cells into
cardiomyocytes. Circ Res 2002;91:189–201.
[7] Bader A, Al-Dubai H, Weitzer G. Leukemia inhibitory factor
modulates cardiogenesis in embryoid bodies in opposite fashion.
Circ Res 2000;86:787–794.
[8] Schuldiner M, Yanuka O, Itskovitz-Eldor J, Melton DA, Benvenisty
N. Effects of eight growth factors on the differentiation of cells
derived from human embryonic stem cells. Proc Natl Acad Sci USA
2000;97:11307–11312.
[9] Gajovic S, St-Onge L, Yokota Y, Gruss P. Retinoic acid mediates
Pax6 expression during in vitro differentiation of embryonic stem
cells. Differentiation 1997;62:187–192.
[10] Jiang Y, Drysdale TA, Evans T. A role for GATA-4 / 5 / 6 in the
regulation of Nkx2.5 expression with implications for patterning of
the precardiac field. Dev Biol 1999;216:57–71.
[11] Dickman ED, Smith SM. Selective regulation of cardiomyocyte
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
gene expression and cardiac morphogenesis by retinoic acid. Dev
Dyn 1996;206:39–48.
Nemer G, Nemer M. Regulation of heart development and function
through combinatorial interactions of transcription factors. Ann Med
2001;33:604–610.
Grepin C, Nemer G, Nemer M. Enhanced cardiogenesis in embryonic stem cells overexpressing the GATA-4 transcription factor.
Development 1997;124:2387–2395.
Morin S, Charron F, Robitaille L, Nemer M. GATA-dependent
recruitment of MEF2 proteins to target promoters. EMBO J
2000;19:2046–2055.
Lin Q, Schwarz J, Bucana C, Olson EN. Control of mouse cardiac
morphogenesis and myogenesis by transcription factor MEF2C.
Science 1997;276:1404–1407.
Monzen K, Shiojima I, Hiroi Y, Kudoh S, Oka T, Takimoto E,
Hayashi D, Hosoda T, Habara-Ohkubo A, Nakaoka T, Fujita T,
Yazaki Y, Komuro I. Bone morphogenetic proteins induce cardiomyocyte differentiation through the mitogen-activated protein
kinase kinase kinase TAK1 and cardiac transcription factors Csx /
Nkx-2.5 and GATA-4. Mol Cell Biol 1999;19:7096–7105.
Maltsev VA, Wobus AM, Rohwedel J, Bader M, Hescheler J.
Cardiomyocytes differentiated in vitro from embryonic stem cells
developmentally express cardiac-specific genes and ionic currents.
Circ Res 1994;75:233–244.
Zhang YM, Hartzell C, Narlow M, Dudley Jr. SC. Stem cell-derived
cardiomyocytes demonstrate arrhythmic potential. Circulation
2002;106:1294–1299.
Sauer H, Rahimi G, Hescheler J, Wartenberg M. Role of reactive
oxygen species and phosphatidylinositol 3-kinase in cardiomyocyte
differentiation of embryonic stem cells. FEBS Lett 2000;476:218–
223.
Finkel T. Reactive oxygen species and signal transduction. IUBMB
Life 2001;52:3–6.
Habara-Ohkubo A. Differentiation of beating cardiac muscle cells
from a derivitive of P19 embryonal carcinoma cells. Cell Struct
Funct 1996;21:101–110.
Monge JC, Stewart DJ, Cernacek P. Differentiation of embryonal
carcinoma cells to a neural or cardiomyocyte lineage is associated
with selective expression of endothelin receptors. J Biol Chem
1995;270:15385–15390.
Paquin J, Danalache BA, Jankowski M, McCann SM, Gutkowska J.
Oxytocin induces differentiation of P19 embryonic stem cells to
cardiomyocytes. Proc Natl Acad Sci USA 2002;99:9550–9555.
Wobus AM, Kleppisch T, Maltsev V, Hescheler J. Cardiomyocytelike cells differentiated in vitro from embryonic carcinoma cells P19
are characterized by functional expression of adrenoceptors and
Ca 21 channels. In Vitro Cell Dev Biol Anim 1994;30A:425–434.
Hosoda T, Monzen K, Hiroi Y, Oka T, Takimoto E, Yazaki Y, Nagai
R, Komuro I. A novel myocyte-specific gene Midori promotes the
differentiation of P19CL6 cells into cardiomyocytes. J Biol Chem
2001;276:35978–35989.
Anversa P, Nadal-Ginard B. Myocyte renewal and ventricular
remodelling. Nature 2002;415:240–243.
Engel FB, Hauck L, Cardoso MC, Leonhardt H, Dietz R, von
Harsdorf R. A mammalian myocardial cell-free system to study cell
cycle reentry in terminally differentiated cardiomyocytes. Circ Res
1999;85:294–301.
Koh GY, Soonpaa MH, Klug MG, Pride HP, Cooper BJ, Zipes DP,
Field LJ. Stable fetal cardiomyocyte grafts in the hearts of
dystrophic mice and dogs. J Clin Invest 1995;96:2034–2042.
Koh GY, Soonpaa MH, Klug MG, Field LJ. Long-term survival of
AT-1 cardiomyocyte grafts in syngeneic myocardium. Am J Physiol
1993;264:H1727–1733.
Etzion S, Battler A, Barbash IM, Cagnano E, Zarin P, Granot Y,
Kedes LH, Kloner RA, Leor J. Influence of embryonic cardiomyocyte transplantation on the progression of heart failure in a
rat model of extensive myocardial infarction. J Mol Cell Cardiol
2001;33:1321–3019.
´
´ / Cardiovascular Research 58 (2003) 369–377
M. J. Goldenthal, J. Marın-Garcıa
[31] Muller-Ehmsen J, Whittaker P, Kloner RA, Dow JS, Sakoda T, Long
TI, Laird PW, Kedes L. Survival and development of neonatal rat
cardiomyocytes transplanted into adult myocardium. J Mol Cell
Cardiol 2002;34:107–116.
[32] Zhang M, Methot D, Poppa V, Fujio Y, Walsh K, Murry CE.
Cardiomyocyte grafting for cardiac repair: graft cell death and
anti-death strategies. J Mol Cell Cardiol 2001;33:907–921.
[33] Reinecke H, Murry CE. Taking the death toll after cardiomyocyte
grafting: a reminder of the importance of quantitative biology. J Mol
Cell Cardiol 2002;34:251–253.
[34] Taylor DA, Atkins BZ, Hungspreugs P, Jones TR, Reedy MC,
Hutcheson KA, Glower DD. Kraus WE: Regenerating functional
myocardium: improved performance after skeletal myoblast transplantation. Nat Med 1998;4:929–933.
[35] Kessler PD. Byrne BJ: Myoblast cell grafting into heart muscle:
cellular biology and potential applications. Annu Rev Physiol
1999;61:219–242.
[36] Robinson SW, Cho PW, Levitsky HI, Olson JL, Hruban RH, Acker
MA, Kessler PD. Arterial delivery of genetically labelled skeletal
myoblasts to the murine heart: long-term survival and phenotypic
modification of implanted myoblasts. Cell Transplant 1996;5:77–91.
[37] Menasche P. Cell transplantation for the treatment of heart failure.
Semin Thorac Cardiovasc Surg 2002;14:157–166.
[38] Jennings RB, Reimer KA. Lethal myocardial ischemic injury. Am J
Pathol 1981;102:241–255.
[39] Murry CE, Wiseman RW, Schwartz SM, Hauschka SD. Skeletal
myoblast transplantation for repair of myocardial necrosis. J Clin
Invest 1996;98:2512–2523.
[40] Jackson KA, Mi T, Goodell MA. Hematopoietic potential of stem
cells isolated from murine skeletal muscle. Proc Nat Acad Sci USA
1999;96:14482–14486.
[41] Reinecke H, Poppa V, Murry CE. Skeletal muscle stem cells do not
trans-differentiate into cardiomyocytes after cardiac grafting. J Mol
Cell Cardiol 2002;34:241–249.
[42] Hakuno D, Fukuda K, Makino S, Konishi F, Tomita Y, Manabe T,
Suzuki Y, Umezawa A, Ogawa S. Bone marrow-derived regenerated
cardiomyocytes express functional adrenergic and muscarinic receptors. Circulation 2002;105:380–386.
[43] Tomita S, Li RK, Weisel RD, Mickle DA, Kim EJ, Sakai T, Jia ZQ.
Autologous transplantation of bone marrow cells improves damaged
heart function. Circulation 1999;100:II247–256.
[44] Orlic D, Kajstura J, Chimenti S, Jakoniuk I, Anderson SM, Li B,
Pickel J, McKay R, Nadal-Ginard B, Bodine DM, Leri A, Anversa
P. Bone marrow cells regenerate infarcted myocardium. Nature
2001;410:701–705.
[45] Jackson KA, Majka SM, Wang H, Pocius J, Hartley CJ, Majesky
MW, Entman ML, Michael LH, Hirschi KK, Goodell MA. Regeneration of ischemic cardiac muscle and vascular endothelium by adult
stem cells. J Clin Invest 2001;107:1395–1402.
[46] Wang JS, Shum-Tim D, Galipeau J, Chedrawy E, Eliopoulos N,
Chiu RC. Marrow stromal cells for cellular cardiomyoplasty:
feasibility and potential clinical advantages. J Thorac Cardiovasc
Surg 2000;120:999–1005.
[47] Makino S, Fukuda K, Miyoshi S, Konishi F, Kodama H, Pan J, Sano
M, Takahashi T, Hori S, Abe H, Hata J, Umezawa A, Ogawa S.
Cardiomyocytes can be generated from marrow stromal cells in
vitro. J Clin Invest 1999;103:697–705.
377
[48] Bittner RE, Schofer C, Weipoltshammer K, Ivanova S, Streubel B,
Hauser E, Freilinger M, Hoger H, Elbe-Burger A, Wachtler F.
Recruitment of bone-marrow-derived cells by skeletal and cardiac
muscle in adult dystrophic mdx mice. Anat Embryol (Berl)
1999;199:391–396.
[49] Toma C, Pittenger MF, Cahill KS, Byrne BJ, Kessler PD. Human
mesenchymal stem cells differentiate to a cardiomyocyte phenotype
in the adult murine heart. Circulation 2002;105:93–98.
[50] Liechty KW, MacKenzie TC, Shaaban AF, Radu A, Moseley AM,
Deans R, Marshak DR, Flake AW. Human mesenchymal stem cells
engraft and demonstrate site-specific differentiation after in utero
transplantation in sheep. Nat Med 2000;6:1282–1286.
[51] Laflamme MA, Myerson D, Saffitz JE, Murry CE. Evidence for
cardiomyocyte repopulation by extracardiac progenitors in transplanted human hearts. Circ Res 2002;90:634–640.
[52] Quaini F, Urbanek K, Beltrami AP, Finato N, Beltrami CA, NadalGinard B, Kajstura J, Leri A, Anversa P. Chimerism of the
transplanted heart. New Engl J Med 2002;346:5–15.
[53] Terada N, Hamazaki T, Oka M, Hoki M, Mastalerz DM, Nakano Y,
Meyer EM, Morel L, Petersen BE, Scott EW. Bone marrow cells
adopt the phenotype of other cells by spontaneous cell fusion.
Nature 2002;416:542–545.
[54] Condorelli G, Borello U, De Angelis L, Latronico M, Sirabella D,
Coletta M, Galli R, Balconi G, Follenzi A, Frati G, Cusella De
Angelis MG, Gioglio L, Amuchastegui S, Adorini L, Naldini L,
Vescovi A, Dejana E, Cossu G. Cardiomyocytes induce endothelial
cells to trans-differentiate into cardiac muscle: implications for
myocardium regeneration. Proc Natl Acad Sci USA 2001;98:10733–
10738.
[55] Clarke DL, Johansson C, Wilbertz J, Veress B, Nilsson E, Karlstrom
H, Lendahl U, Frisen J. Generalized potential of adult neural stem
cells. Science 2000;288:1660–1663.
[56] Marin-Garcia J, Goldenthal MJ. Understanding the impact of
mitochondrial defects in cardiovascular disease. A review. J Card
Fail 2002;8:347–361.
[57] Zullo SJ. Gene therapy of mitochondrial DNA mutations: a brief,
biased history of allotopic expression in mammalian cells. Semin
Neurol 2001;21:327–335.
[58] Bremer S, Worth AP et al. Establishment of an in vitro reporter gene
assay for developmental cardiac toxicity. Toxicol Vitro
2001;15:215–223.
[59] Scholz G, Pohl I, Genschow E, Klemm M, Spielmann H. Embryotoxicity screening using embryonic stem cells in vitro: correlation to in vivo teratogenicity. Cells Tissues Organs 1999;165:203–
211.
[60] Rohwedel J, Guan K, Hegert C, Wobus AM. Embryonic stem cells
as an in vitro model for mutagenicity, cytoxicity and embryotoxicity
studies: present state and future prospects. Toxicol Vitro
2001;15:741–753.
[61] Odorico JS, Kaufman DS, Thomson JA. Multilineage differentiation
from human embryonic stem cell lines. Stem Cells 2001;19:193–
204.
[62] Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD,
Ortiz-Gonzalez XR, Reyes M, Lenvik T, Lund T, Blackstad M, Du
J, Aldrich S, Lisberg A, Low WC, Largaespada DA, Verfaillie CM.
Pluripotency of mesenchymal stem cells derived from adult marrow.
Nature 2002;418:41–49.